Nonspecific hydridization inhibitors, clinical examination reagents and clinical examination method

ABSTRACT

The present invention relates to an inhibitor capable of increasing accuracy of the reaction in hybridization, and inhibiting nonspecific hybridization, a clinical diagnostic reagent, and a method of clinical analysis whereby nonspecific hybridization in clinical analysis is inhibited, and a target nucleic substance is detected easily with high accuracy. The inhibitor and the clinical diagnostic reagent contains polymer (H) having a nonspecific hybridization inhibitory action. The polymer has in its molecule at least one of carboxyl and sulfone groups, and phosphorylcholine-like groups, and has a weight average molecular weight of 1000 to 5000000. The method of clinical analysis includes contacting a sample with a test agent capable of hybridizing with a specific nucleic substance in the presence of the inhibitor.

FIELD OF ART

[0001] The present invention relates to inhibitors for inhibitingnonspecific hybridization in detecting a target nucleic substance in asample in the field of clinical analysis and the like, clinicaldiagnostic reagents containing the inhibitor, and a method of clinicalanalysis using the inhibitor.

BACKGROUND ART

[0002] In the art of clinical analysis, detection of nucleic substancessuch as DNAs and RNAs is made through hybridization. This methodinvolves bringing a labeled sample into contact with a solid phase onwhich a DNA or the like of a sequence complementary to the targetnucleic substance is fixed, washing away other materials than the targetnucleic substance, and measuring the activity of the labeled materialbound to the solid phase.

[0003] In the field of clinical analysis, by detecting a target nucleicsubstance through hybridization, the sequence of the nucleic substanceis required to be recognized precisely.

[0004] In evaluating a sample by hybridization, it is conventionally andwidely known to add surface active agents such as sodium dodecyl sulfate(abbreviated as SDS) or N-lauroyl sarcocine (abbreviated as N-LS); orproteins such as bovine serum albumin (abbreviated as BSA) or casein,for inhibiting nonspecific hybridization. However, such surface activeagents and proteins have little nonspecific hybridization inhibitoryaction, so that the sequence of the nucleic substance cannot berecognized precisely.

[0005] Patrick et al. (Nature Biotechnology, 17, 365-370 (1999)) proposeto inhibit binding of SNPs of a DNA by applying electrical current onthe surface of a DNA chip. This method, however, requires dedicated DNAchips, and cannot be applied to every existing DNA chip.

[0006] On the other hand, polymers having a phosphorylcholine-like groupare under examination for their possible use in the field of clinicalanalysis. Polymers having a phosphorylcholine-like group are known tohave excellent biocompatibilities such as blood compatibility, abilityto inactivate complements, and nonadsorbability of biomaterials, as wellas excellent antifouling property and moisture holding property, due totheir structures similar to phosphlipids originated from biomembrane.Researches and developments in synthesis and use of the polymers havebeen actively made for developing bio-related materials that make themost of these properties. JP-7-5177-A, JP-7-83923-A, and JP-10-114800-Adisclose highly accurate clinical analysis techniques in which polymershaving a phosphorylcholine group inhibit adsorption of proteins by avessel.

[0007] However, it is not known that the sequence of a nucleic substancemay be recognized precisely, i.e., nonspecific hybridization may beinhibited, by using particular polymers having phosphorylcholine groups.

DISCLOSURE OF THE INVENTION

[0008] It is an object of the present invention to provide a nonspecifichybridization inhibitor that increases accuracy of the reaction inhybridization, and inhibits nonspecific hybridization.

[0009] It is another object of the present invention to provide aclinical diagnostic reagent that inhibits nonspecific hybridizationduring hybridization for clinical analysis, and enables detection of atarget nucleic substance with high efficiency and accuracy.

[0010] It is yet another object of the present invention to provide amethod of clinical analysis whereby nonspecific hybridization inclinical analysis is inhibited, and a target nucleic substance isdetected easily with high accuracy.

[0011] According to the present invention, there is provided anonspecific hybridization inhibitor comprising a polymer (H) having anonspecific hybridization inhibitory action, said polymer having in itsmolecule at least one of carboxyl and sulfone groups, andphosphorylcholine-like groups, and having a weight average molecularweight of 1000 to 5000000.

[0012] According to the present invention, there is also provided aclinical diagnostic reagent comprising the above inhibitor and a testagent.

[0013] According to the present invention, there is further provided amethod of clinical analysis comprising the steps of:

[0014] (1) contacting a sample with a test agent capable of hybridizingwith a specific nucleic substance in the presence of the above inhibitorunder particular conditions for hybridizing said specific nucleicsubstance with said test agent, and

[0015] (2) detecting a reactant generated by hybridization with saidtest agent in step (1).

PREFERRED EMBODIMENT OF THE INVENTION

[0016] The inhibitor according to the present invention contains apolymer (H) having a nonspecific hybridization inhibitory action, whichpolymer has in its molecule at least one of carboxyl and sulfone groups,and phosphorylcholine-like groups (abbreviated as PC group), and has aparticular weight average molecular weight.

[0017] In the polymer (H), the ratio of the at least one of carboxyl andsulfone groups to the PC groups is not particularly limited as long asthe polymer (H) has the nonspecific hybridization inhibitory action, andmay suitably be decided depending on the kinds and ratio of the monomersused in preparing the polymer (H) to be discussed later, or themolecular weight of the polymer (H).

[0018] The polymer (H) may optionally have other groups as long as thepolymer (H) has the essential groups as mentioned above, has theparticular molecular weight, and has the nonspecific hybridizationinhibitory action. Examples of such other groups may include variousgroups such as phosphoric acid, betaine, primary amino, secondary amino,tertiary amino, quaternary ammonium, acrylamide, methacrylamide,4-butane lactam(2-pyrrolidone), 4-butane lactim(2-hydroxy-1-pyrroline),6-hexane lactam(ε-caprolactam), polyethylene oxide, polypropylene oxide,and block or random polymers of polyethylene oxide and polypropyleneoxide. The examples of the other groups may also include groups derivedfrom various hydrophobic monomers to be discussed later.

[0019] The content of such other groups in the polymer (H) may suitablybe selected from the range of content that will not disturb or willenhance the objects of the present invention.

[0020] The molecular weight of the polymer (H) is usually 1000 to5000000, preferably 10000 to 2000000, by weight average molecularweight. It is not preferred that the molecular weight is less than 1000since sufficient inhibition of the nonspecific hybridization is hard tobe achieved, and that the molecular weight is over 5000000 since theviscosity of the polymer (H) is too high, which may inhibithybridization.

[0021] The polymer (H) may be prepared, for example, by polymerizing amonomer having a PC group (referred to as PC monomer) and a monomerhaving a sulfone group (referred to as SA monomer) and/or a monomerhaving a carboxyl group (referred to as CA monomer), and optionallyother monomers such as a hydrophobic monomer as needed, by conventionalradical polymerization. Each monomer used in the polymerization does nothave to be of a single kind, but may be of two or more kinds.

[0022] Examples of the PC monomer may include monomers represented bythe formula (1):

[0023] In the formula (1), X stands for a divalent organic residue, Ystands for an alkyleneoxy group having 1 to 6 carbon atoms, and Z standsfor a hydrogen atom or R⁵O (C═O)—, wherein R⁵ stands for an alkyl orhydroxyalkyl group having 1 to 10 carbon atoms. R¹ in the formula (1)stands for a hydrogen atom or a methyl group, and R², R³, and R⁴ are thesame or different groups, and each stands for a hydrogen atom, or analkyl or hydroxyalkyl group having 1 to 6 carbon atoms. m is 0 or 1, andn denotes an integer of 1 to 4.

[0024] X in the formula (1) may be, for example, —C₆H₄—, —C₆H₁O—,—(C═O)O—, —O—, —CH₂O—, —(C═O)NH—, —O(C═O)—, —O(C═O)O—, —C₆H₄O—,—C₆H₄CH₂O—, or —C₆H₄(C═O)O—. Y in the formula (1) may be, for example, amethyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, or hexyloxy group.R⁵ in the formula for Z may be, for example, an alkyl group having 1 to10 carbon atoms, such as a methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, or decyl group; or a hydroxyalkyl group having 1to 10 carbon atoms, such as a hydroxymethyl, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxypropyl, 4-hydroxybutyl, 2-hydroxybutyl,5-hydroxypentyl, 2-hydroxypentyl, 6-hydroxyhexyl, 2-hydroxyhexyl,7-hydroxyheptyl, 2-hydroxyheptyl, 8-hydroxyoctyl, 2-hydroxyoctyl,9-hydroxynonyl, 2-hydroxynonyl, 10-hydroxydecyl, or 2-hydroxydecylgroup.

[0025] Examples of the PC monomer may include2-((meth)acryloyloxy)ethyl-2′-(trimethylammonio)ethyl phosphate,3-((meth)acryloyloxy)propyl-2′-(trimethylammonio)ethyl phosphate,4-((meth)acryloyloxy)butyl-2′-(trimethylammonio)ethyl phosphate,5-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethyl phosphate,6-((meth)acryloyloxy)hexyl-2′-(trimethylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(triethylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(tripropylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(tributylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(tricyclohexylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(triphenylammonio)ethyl phosphate,2-((meth)acryloyloxy)ethyl-2′-(trimethanol ammonio)ethyl phosphate,2-((meth)acryloyloxy)propyl-2′-(trimethylammonio)ethyl phosphate,2-((meth)acryloyloxy)butyl-2′-(trimethylammonio)ethyl phosphate,2-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethyl phosphate,2-((meth)acryloyloxy)hexyl-2′-(trimethylammonio)ethyl phosphate,2-(vinyloxy) ethyl-2′-(trimethylammonio)ethyl phosphate,2-(allyloxy)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(p-vinylbenzyloxy)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(p-vinylbenzoyloxy)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(styryloxy)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(p-vinylbenzyl)ethyl-2′-(trimethylammonio) ethyl phosphate,2-(vinyloxycarbonyl)ethyl-2′-(trimethylanmmonio)ethyl phosphate,2-(allyloxycarbonyl)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(acryloylamino)ethyl-2′-(trimethylammonio)ethyl phosphate,2-(vinylcarbonylamino)ethyl-2′-(trimethylammonio)ethyl phosphate,ethyl-(2′-trimethylammonioethylphosphorylethyl) fumarate,butyl-(2′-trimethylammonioethylphophorylethyl) fumarate,hydroxyethyl-(2′-trimethylammonioethylphosphorylethyl) fumarate,ethyl-(2′-trimethylammonioethylphosphorylethyl) maleate,butyl-(2′-trimethylammonioethylphosphorylethyl) maleate, andhydroxyethyl-(2′-trimethylammonioethylphosphorylethyl) maleate.

[0026] Among these examples,2-((meth)acryloyloxy)ethyl-2′-(trimethylammonio) ethyl phosphate ispreferred, and in particular2-(methacryloyloxy)ethyl-2′-(trimethylammonio)ethyl phosphate (i.e.,2-(methacryloyloxy)ethylphosphorylcholine) (abbreviated as MPC) ispreferred for its availability. As used herein, (meth)acryloyl meansmethacryloyl and/or acryloyl.

[0027] The PC monomer may be prepared by a conventional method, forexample, by reacting 2-hydroxyethyl methacrylate and2-bromoethylphosphoryl dichloride in the presence of a tertiary base, ofwhich reaction product is further reacted with a tertiary amine, asdisclosed in JP-54-63025-A, or by reacting a polymerizable monomerhaving a hydroxyl group and a cyclic phosphorus compound, followed byring-opening with a tertiary amine, as disclosed in JP-58-154591-A.

[0028] Examples of the SA monomer may include 2-methylpropane sulfonate,2-dimethylpropane sulfonate, styrene sulfonate, and 2-sulfoethylmethacrylate. Among these examples, 2-methylpropane sulfonate,2-dimethylpropane sulfonate, and styrene sulfonate are preferred.

[0029] Examples of the CA monomer may include (meth) acrylic acid,3-pentenoic acid, 4-pentenoic acid, 3-acryloxypropionic acid,2-(meth)acryloyloxyethylphthalic acid. Among these examples, methacrylicacid and acrylic acid are preferred.

[0030] The hydrophobic monomer may be a monomer represented by theformula (2):

[0031] In the formula (2), R⁶ stands for a hydrogen atom or a methylgroup, L¹ stands for —C₆H₄—, —C₆H₁₀—, —(C═O)O—, —O—, —(C═O)NH—,—O(C═O)—, or —O(C═O)O—, and L² stands for a hydrogen atom, —(CH₂)g-L³,or ((CH₂)_(p)—O)_(h)-L³, wherein g and h each denotes an integer of 1 to24, p denotes an integer of 3 to 5, and L³ stands for a hydrogen atom, amethyl group, —C₆H₅, or —OC₆H₅.

[0032] Examples of the hydrophobic monomer may include straight orbranched alkyl (meth)acrylates, such as methyl (meth)acrylate, ethyl(meth)acrylate, butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate,lauryl (meth)acrylate, or stearyl (meth)acrylate; cyclic alkyl(meth)acrylates such as cyclohexyl (meth)acrylate; aromatic ring(meth)acrylates such as benzyl (meth)acrylate or phenoxyethyl(meth)acrylate; polyalkylene glycol (meth) acrylates such aspolypropylene glycol (meth)acrylate; styrene monomers such as styrene,methylstyrene, or chloromethylstyrene; vinyl ether monomers such asmethylvinyl ether or butylvinyl ether; and vinyl ester monomers such asvinyl acetate or vinyl propionate. Among these examples, butylmethacrylate and lauryl methacrylate are preferred.

[0033] The polymer (H) may be a polymer having the particular molecularweight mentioned above and obtained by polymerizing a monomercomposition containing 5 to 95 mol %, preferably 10 to 90 mol % of thePC monomer and 5 to 95 mol %, preferably 10 to 90 mol % of at least oneof the SA and CA monomers, or a monomer composition containing 5 to 90mol %, preferably 10 to 85 mol % of the PC monomer, 5 to 90 mol %,preferably 10 to 85 mol % of at least one of the SA and CA monomers, and5 to 60 mol %, preferably 5 to 50 mol % of the hydrophobic monomer.

[0034] As used herein, the phrase “at least one of the SA and CAmonomers” includes all the embodiments wherein either the SA or CAmonomer is contained alone, and wherein both the SA and CA monomers arecontained together. Further, each of the monomer compositions mentionedabove may optionally contain other monomers, in addition to the PCmonomer, the SA monomer, the CA monomer, and the hydrophobic monomer. Ineach of the monomer compositions, the content of such other monomers isusually not higher than 10 mol %, preferably 5 mol %. In someembodiments, it is rather preferred not to contain such other monomers.

[0035] If the contents of the PC monomer and at least one of the SA andCA monomers in each monomer composition are all less than 5 mol %, thenonspecific hybridization cannot be inhibited sufficiently, thus notbeing preferred. The polymer (H) may be prepared by conventional radicalcopolymerization.

[0036] There is no particular limitation imposed on the presentinhibitor as long as the inhibitor contains the polymer (H). The presentinhibitor may be used by adding the inhibitor to the hybridizationsystem to mix so that the content of the polymer (H) in thehybridization system is usually 0.0001 to 20 wt %, preferably 0.001 to10 wt %. If the content of the polymer (H) is less than 0.0001 wt %, thenonspecific hybridization inhibitory action may not be achievedsufficiently, whereas if over 20 wt %, the viscosity of thehybridization system is too high, which may inhibit the hybridizationreaction, thus not being preferred.

[0037] The inhibitor of the present invention may optionally containother components, such as surface active agents, solvents, andpreservatives, as long as the effects of the present invention are notimpaired. The inhibitor may be in the form of powders or a solution, butthe solution form is preferred in use.

[0038] The clinical diagnostic reagent of the present invention is atest agent kit including the present inhibitor and a test agent. Thetest agent may be any nucleic substance as long as it hybridizes withthe particular nucleic substance to be detected, and may be, forexample, a test agent used in conventional clinical diagnostic reagentsdepending on the target.

[0039] As used herein, the nucleic substance means any one or moregroups of compounds containing carbohydrate molecules, such as pentoseor hexose, having one or more phosphorus molecules bound thereto, eachof which carbohydrate molecules binds to a base such as adenine derivedfrom purine or thymine derived from pyrimidine. Specifically, naturallyoccurring nucleic acid molecules include genomic DNAs, genomic RNAs, andvarious types of RNAs, such as mRNAs, tRNAs, and rRNAs, and also cDNAs.Further, the nucleic substance also includes synthesized DNAs or hybridsof naturally occurring DNAs and synthesized DNAs.

[0040] The nucleic substance may be either single or double stranded,linear or circular, or plasmids, or oligonucleotide or polynucleotide.The nucleic substance may include bases or base pairs having from about10 to about 20000 bases (20 kb). In addition to such naturally occurringsubstances, for example, those obtained from such sources as ATCC orGene Bank (GenBank) Libraries, and synthetic compositions, i.e., nucleicacid-like materials, may also be included. Synthetic nucleic acids maybe prepared by a variety of solution or solid phase methods. Generally,solid phase synthesis is preferred. Synthesis of nucleic acids byphosphitetriester, phosphotriester, and H-phosphonate chemistries arewidely available. (Itakura, U.S. Pat. No. 4,458,066 and U.S. Pat. No.4,500,707; Garuthers et al., Genetic Engineering, 4:1-17 (1982); Jones,chapter 2, Atkinson, et al., chapter 3, and Sproat, et al., chapter 4,in Oligonucleotide Synthesis: A Practical Approach, Gait (ed.), IRLPress, Washington, D.C. (1984)).

[0041] In the clinical diagnostic reagent of the present invention, thecontent of the present inhibitor may suitably be selected depending onthe method of clinical analysis and the target to be detected, and maybe such that the content of the polymer (H) of the inhibitor in thehybridization system is usually 0.0001 to 20 wt %, preferably 0.001 to10 wt %.

[0042] The clinical diagnostic reagent of the present invention mayoptionally contain other compounds required for the test agent, such asvarious additives and solvents usually contained in clinical diagnosticreagents. The usage of the clinical diagnostic reagent is notparticularly limited, and the diagnostic reagent may be used, forexample, with reference to the method of clinical analysis according tothe present invention to be discussed later.

[0043] The method of clinical analysis according to the presentinvention includes step (1) of first contacting a sample with a testagent capable of hybridizing with a specific nucleic substance, in thepresence of the inhibitor of the present invention under the particularconditions for hybridizing the specific nucleic substance with the testagent.

[0044] Step (1) may be performed using the inhibitor of the presentinvention or the clinical diagnostic reagent of the present invention.Step (1) may be performed, for example, by adding the present inhibitorto a system containing a sample and a test agent, and keeping the systemunder the particular conditions; by keeping a system containing a sampleand a test agent under the particular conditions, and adding the presentinhibitor to the system; or by adding the present inhibitor to a sampleand/or a test agent in advance, and then contacting the sample and thetest agent under the particular conditions.

[0045] Step (1) may be carried out, for example, by adding the presentinhibitor upon or after fixing the test agent on a carrier, andhybridizing the test agent with a sample. In this case, the inhibitor ofthe present invention may be in the form of either a solution orpowders, and may preferably be in a solution form for its readiness ofhomogenization with the reaction system.

[0046] The carrier may be, for example, a membrane such as anitrocellulose, nylon, or PVDF membrane; or a plate or a flat plate suchas those made of polystyrene, polypropylene, glass, or metal, but notlimited to these examples. The test agent may be fixed to the carrierfor example by electrostatic binding, by using ionicity, by covalentbonding by UV rays or the like, or by covalent bonding using acrosslinking reagent.

[0047] Alternatively, step (1) may also be performed in a solutionwithout the carrier. Examples of such method may include polymerasechain reaction (PCR), sequence reaction, RNA-dependent DNA synthesisusing a reverse transcriptase, or various transcription reactions usinga DNA polymerase employed in gene engineering.

[0048] In step (1), the particular conditions for hybridizing thespecific nucleic substance with the test agent may suitably be selected,for example, from suitable conditions for Southern hybridization whenthe specific nucleic substance to be detected is a DNA, or from suitableconditions for Northern hybridization when the specific nucleicsubstance to be detected is an RNA.

[0049] The method of clinical analysis according to the presentinvention includes step (2) of detecting a reactant generated byhybridization with the test agent in step (1).

[0050] In step (2), the reactant may be detected, for example, bylabeling in accordance with a conventional labeling technique fordetecting a reactant, and measuring the activity or the like of thelabel. The labeling may be performed with radioisotopes such as ³²P,¹⁴C, or ³H, fluorescent dyes, enzyme proteins, or antibodies to variousantigenic substances. The labeled compounds may be prepared by using aDNA polymerase with the labeling reagent as the substrate, or bychemical reactions. The method of detecting the reactant may suitably beselected depending on the labeling method. For example, when thelabeling is performed with a radioisotope, the labeled reactant may bedetected by means of a scintillation counter or auto radiography; whenlabeled with a fluorescent dye, by means of a detector utilizing lasers;and when labeled with an enzyme protein or various antigens, by means ofdetection of chemiluminescence, fluorescence, or coloring of the enzymesubstrate.

[0051] The method of clinical analysis according to the presentinvention is not particularly limited as long as the method includessteps (1) and (2) discussed above, and may easily be practiced utilizingvarious conventional methods. An embodiment utilizing Southern blothybridization is discussed below. The following discussion does notinclude the description of the inhibitor of the present invention, butthe method of clinical analysis according to the present invention maybe performed by suitably adding the present inhibitor at various timepoints and in the preferred amount mentioned above.

[0052] First, from biological tissues such as blood, genomic DNAs areprepared with ISOGEN (manufactured by NIPPON GENE CO., LTD.). Theresulting DNAs are separated by 0.7% agarose gel electrophoresis. Afterthe completion of the electrophoresis, the gel is immersed in a 0.25 Nhydrochloric acid solution at room temperature for 15 minutes, and thenin a 0.4 N sodium hydroxide solution at room temperature for 30 minutesunder gentle shaking. On a blot table previously prepared, the resultinggel, a Gene Screen Plus membrane (manufactured by DuPont) , 3 MM filterpapers (manufactured by Whatman plc), Kimtowels, and a weight are placedon top of another in this order, and left overnight to transcript theDNAs in the gel to the Gene Screen Plus membrane. When the transcriptionis completed, the Gene Screen Plus membrane is immersed in a 0.5 M Trisbuffer at room temperature for 15 minutes, dried at room temperature,and baked at 80° C. for 2 hours. The resulting membrane is shaken in 2×SSC (0.3 M sodium chloride, 0.03 M sodium citrate), and then immersed ina rapid hybridization buffer (manufactured by Amersham PharmaciaBiotech) at 65° C. for 15 minutes under shaking for pre-hybridization. Asolution of target DNA probes (³²P labeled) previously labeled with[γ³²P]-dCTP by nick translation is added to the hybridization buffer,and the hybridization is effected at 65° C. for 2 hours. The membrane isshaken in 0.1× SSC (0.015 M sodium chloride, 0.0015 M sodium citrate) at65° C. for 15 minutes. This operation is repeated twice to wash awayexcess probes. Excess moisture is removed on filter papers, and X-rayfilm is exposed to the radioactive rays from the probes at −70° C. for24 hours, and developed. The objective bands are quantified by adensitometer. (Molecular Cloning: A LABORATORY MANUAL, 2nd edition, ColdSpring Harbor Laboratory Press (1989)).

EXAMPLES

[0053] The present invention will now be explained in more detail withreference to Examples and Comparative Examples, but the presentinvention is not limited thereto.

Synthesis Example 1

[0054] 19.4 g of MPC and 0.6 g of methacrylic acid (abbreviated as MA)were dissolved in 40 g of water, and poured into a four neck flask. Thesolution was bubbled with nitrogen for 30 minutes, 1.6 g of succinicperoxide was added at 60° C., and the mixture was polymerized for 8hours. The polymerization solution was added dropwise to 3 L of acetoneunder stirring. The resulting precipitate was taken out by filtration,and vacuum dried at room temperature for 48 hours, to obtain 14.6 g ofpolymer powders (abbreviated as Polymer (P1)). The molecular weight ofPolymer (P1) was analyzed by gel permeation chromatography (GPC),wherein the elution solvent was a 20 mM phosphate buffer (pH 7.4), thereference material was polyethylene glycol, and the detection was madein accordance with the refractive index. Further, the compositionalratio of and the groups contained in Polymer (P1) were determined by¹H-NMR. The results are shown in Table 1.

Synthesis Examples 2 to 5

[0055] Polymer powders were prepared in the same way as in SynthesisExample 1, except that the kinds and the compositional ratio of themonomers used in Synthesis Example 1 were changed as shown in Table 1.The obtained polymer powders were referred to as Polymers (P2) to (P5).The same analyses as in Synthesis Example 1 were performed. The resultsare shown in Table 1.

Synthesis Examples 6 and 7

[0056] Polymer powders were prepared in the same way as in SynthesisExample 1, except that the kinds and the compositional ratio of themonomers used in Synthesis Example 1 were changed as shown in Table 1,40 g of water was replaced with 120 g of isopropanol, and 1.6 g ofsuccinic peroxide was replaced with 0.7 g of azobis isobutyronitrile(abbreviated as AIBN). The obtained polymer powders were referred to asPolymers (P6) and (P7). The same analyses as in Synthesis Example 1 wereperformed. The results are shown in Table 1, wherein BMA is anabbreviation for butyl methacrylate.

Synthesis Examples 8 to 10

[0057] Polymer powders were prepared in the same way as in SynthesisExample 1, except that the kinds and the compositional ratio of themonomers used in Synthesis Example 1 were changed as shown in Table 2,and the amount of succinic peroxide was 0.65 g. The obtained polymerpowders were referred to as Polymers (P8) to (P10). The same analyses asin Syntheses Example 1 were performed. The results are shown in Table 2,wherein MPs is an abbreviation for 2-methylpropanesulfonic acid.

Synthesis Examples 11 to 12

[0058] Polymer powders were prepared in the same way as in SynthesisExample 1, except that the kinds and the compositional ratio of themonomers used in Synthesis Example 1 were changed as shown in Table 2,the amount of water was 120 g, and the amount of succinic peroxide was0.23 g. The obtained polymer powders were referred to as Polymers (P11)to (P12). The same analyses as in Synthesis Example 1 were performed.The results are shown in Table 2, wherein Am is an abbreviation foracrylamide, Vp for N-vinyl-2-pyrrolidone, and MANa for sodiummethacrylate. TABLE 1 Synthesis Synthesis Synthesis Synthesis SynthesisSynthesis Synthesis Example 1 Example 2 Example 3 Example 4 Example 5Example 6 Example 7 Kind of Polymer (P1) (P2) (P3) (P4) (P5) (P6) (P7)Monomer (A) MPC 19.4 g 17.1 g 15.5 g 12.0 g 20.0 g 9.79 g 12.3 gComposition (B) MA  0.6 g  2.2 g  4.5 g  8.0 g — 2.85 g 1.79 g (C) BMA —— — — — 2.35 g 5.92 g Molar Ratio of Monomers A/B = 90/10 A/B = 70/30A/B = 50/50 A/B = 30/70 A/B = 100/0 A/B/C = 40/40/20 A/B/C = 40/20/40Polymer Compositional Ratio A/B = 89/11 A/B = 68/32 A/B = 49/51 A/B =29/71 A/B = 100/0 A/B/C = 41/40/19 A/B/C = 40/22/38 Kinds of Groups—PC—COOH —PC—COOH —PC—COOH —PC—COOH —PC —PC—COOH —PC—COOH ContainedWeight Average 153000 550000 1104000 653000 1030000 363000 56000Molecular Weight

[0059] TABLE 2 Synthesis Synthesis Synthesis Synthesis Synthesis Example8 Example 9 Example 10 Example 11 Example 12 Kind of Polymer (P8) (P9)(P10) (P11) (P12) Monomer (A) MPC 23.06 g 17.62 g 11.37 g — —Composition Am — — — 19.75 g — Vp — — — — 22.52 g E4 — — — — — (B) MPs 6.94 g 12.38 g 18.63 g — — MANa — — — 10.25 g  7.48 g Molar Ratio ofMonomers A/B = 70/30 A/B = 50/50 A/B = 30/70 A/B = 70/30 A/B = 70/30Polymer Compositional Ratio A/B = 70/30 A/B = 48/52 A/B = 33/67 A/B =66/34 A/B = 71/29 Kinds of Groups —PC—SO₃H —PC—SO₃H —PC—SO₃H —COONa-—COONa- Contained amide pyrrolidone Weight Average 622000 10700001016000 1197000 800000 Molecular Weight

Example 1-1

[0060] An oligo DNA having the 5′-terminal modified with an amino group(abbreviated as PUC-NH₂) for immobilization, a full match oligo DNAhaving the 5′-terminal modified with biotin and having a sequencecomplementary to PUC-NH₂ (abbreviated as PUC), and mismatch oligo DNAshaving the 5′-terminal modified with biotin, which are SNPs of PUC,(abbreviated as PUC′ and PUC″), each having the DNA sequence shownbelow, were used. PUC-NH₂, PUC, PUC′, and PUC″ are manufactured by ESPECOLIGO SERVICE CORP.

[0061] PUC-NH₂:5′-ACTGGCCGTCGTTTTACAACGTCGTGACTGGG-3′

[0062] PUC:5′-CCCAGTCACGACGTTGTAAA-3′

[0063] PUC′:5′-CCCAGTCACCACGTTGTAAA-3′

[0064] PUC″:5′-CCCAGTCACGTCGTTGTAAA-3′

[0065] 100 μl/well of a 25 pmol/ml PUC-NH₂ solution in a 50 mM phosphatebuffer (pH 8.5) mixed with 1 mM EDTA (abbreviated as E-NaPB) wasdispensed into a DNA-BIND 96-well plate (manufactured by CorningIncorporated), and incubated overnight at 4° C. (immobilization ofPUC-NH₂) Then each well was cleared of the PUC-NH₂ solution, and washedthree times with Dulceco's PBS (abbreviated as D-PBS). 200 μl/well ofthe E-NaPB solution mixed with 3% BSA (abbreviated as B-E-NaPB) wasadded, and incubated at 37° C. for 1 hour (blocking operation-1). Theneach well was cleared of the B-E-NaPB to prepare a PUC-NH₂-immobilizedplate.

[0066] In the PUC-NH₂-immobilized plate, 3.2 wt % of Polymer (P1), 0.2pmol/ml each of PUC, PUC′, and PUC″, and a 75 mM sodium citrate solutioncontaining 750 mM NaCl (abbreviated as 5× SSC) were added in an amountof 100 μl/well, and incubated at 55° C. for 1 hour for hybridization.Each well was cleared of the solution, and washed twice with a 30 mMsodium citrate solution containing 300 mM NaCl (abbreviated as 2× SSC)at 55° C. 200 μl/well of the B-E-NaPB was added, and incubated at 37° C.for 30 minutes (blocking operation-2), and then each well was cleared ofthe B-E-NaPB. 100 μl/well of an avidin-horseradish peroxidase(manufactured by SIGMA-ALDRICH CO.) solution diluted 10000 times withthe B-E-NaPB (abbreviated as Avdin-HRP) was added, and incubated at 37°C. for 30 minutes (avidin-biotin reaction). Each well was then washedthree times with D-PBS, 100 μl/well of the substrate solution includedin the HRP kit was added, and the mixture was incubated at 25° C. for 10minutes (POD-substrate reaction). Subsequently, 100 μl/well of thereaction terminator included in the HRP kit was added, and theabsorbance at 450 nm was measured by means of SPECTRAMAX250 (microplatereader, manufactured by MOLECULAR DEVICES CORPORATION). Incidentally,the HRP kit used herein was Coloring Kit T for Peroxidase (manufacturedby SUMITOMO BAKELITE CO., LTD.).

[0067] From the results of the measurements, the ratios of (absorbanceof PUC)/(absorbance of PUC′) and (absorbance of PUC)/(absorbance ofPUC″) were determined. The results are shown in Table 3.

Examples 1-2 to 1-9 and Comparative Examples 1-1 to 1-2

[0068] Measurements were made in the same way as in Example 1-1, exceptthat Polymer (P1) was replaced with each polymer shown in Table 3. Theresults are shown in Table 3.

Comparative Example 1-3

[0069] Measurements were made in the same way as in Example 1-1, exceptthat the 5× SSC solution containing 3.2 wt % of Polymer (P1) and 0.2pmol/ml each of PUC, PUC′, and PUC″ was replaced with a 5× SSC solutioncontaining 0.2 pmol/ml each of PUC, PUC′, and PUC″. The results areshown in Table 3.

Comparative Example 1-4

[0070] Measurements were made in the same way as in Example 1-1, exceptthat the 5× SSC solution containing 3.2 wt % of Polymer (P1) and 0.2pmol/ml each of PUC, PUC′, and PUC″ was replaced with a 5× SSC solutioncontaining 1.0% casein (manufactured by SIGMA-ALDRICH CO.) and 0.1% N-LS(manufactured by WAKO PURE CHEMICALS INDUSTRIES, LTD.). The results areshown in Table 3.

Comparative Example 1-5

[0071] Measurements were made in the same way as in Example 1-1, exceptthat the 5× SSC solution containing 3.2 wt % of Polymer (P1) and 0.2pmol/ml each of PUC, PUC′, and PUC″ was replaced with 0.2 pmol/ml eachof PUC, PUC′, and PUC″, and a 6× SSC solution containing 10 mg/ml Ficol400 (manufactured by Amersham Pharmacia Biotech, trademark), 10 mg/mlpolyvinylpyrrolidone, 10 mg/ml bovine serum albumin, and 0.5% SDS(Denhard's solution). The results are shown in Table 3. TABLE 3 Kind of(Absorbance of (Absorbance of Polymer PUC)/(Absorbance PUC)/(Absorbance(Abbreviation) of PUC′) of PUC″) Example 1-1 (P1) 4.5 2.0 Example 1-2(P2) 4.0 2.6 Example 1-3 (P3) 2.7 2.4 Example 1-4 (P4) 2.6 2.3 Example1-5 (P6) 4.0 2.7 Example 1-6 (P7) 3.8 2.2 Example 1-7 (P8) 3.1 2.1Example 1-8 (P9) 3.1 2.5 Example 1-9 (P10) 3.2 2.4 Comp. Ex. (P11) 2.41.6 1-1 Comp. Ex. (P12) 2.5 1.8 1-2 Comp. Ex. — 2.0 1.0 1-3 Comp. Ex.Other Inhibitor 1.8 1.0 1-4 Comp. Ex. Other Inhibitor 1.7 1.0 1-5

[0072] From Table 3, it is understood that the ratios of the absorbancein full match (absorbance of PUC) to the absorbance in mismatch(absorbance of PUC′ or PUC″) are higher in Examples than in ComparativeExamples. This means that the inhibitors used in Examples do not inhibitspecific hybridization but inhibit nonspecific hybridization, to realizemore accurate hybridization.

Production Example 1-1

[0073] Into a 96-well multiplate (manufactured by Nalge NuncInternational, white), 150 μl/well of 0.2 mg/mlpoly(lysine-phenylalanine) dissolved in 5× SSC was dispensed and left tostand still at room temperature for 20 hours. The plate was washed witha 50 mM phosphate buffer (pH 8.3). 200 μl/well of 650 μM 2-iminothiolanehydrochloride dissolved in a 200 mM phosphate buffer (pH 8.0) was added,and left to stand still at room temperature for 2 hours. The plate waswashed with a 10 mM phosphate buffer (pH 7.0).

[0074] An oligonucleotide having the 5′-terminal modified with an aminolinker (manufactured by Amersham Pharmacia Biotech) (sequence:5′-CATTAGGGATCCAGCCGTGAATTCGTCACT-3′) for immobilization was dissolvedin a 50 mM phosphate buffer (pH 7.0), and N-succinimidyl4-maleimidobutyrate (abbreviated as GMBS) dissolved in dimethylformamide(abbreviated as DMF) was added in an amount five times the molar amountof the oligonucleotide. The reaction was effected at 37° C. for 90minutes, and the unreacted GMBS was removed to obtain amaleimide-attached oligonucleotide. 25 pmol/well of thismaleimide-attached oligonucleotide was dispensed into a 96-wellmultiplate previously treated with 2-iminothiolane hydrochloride, andreacted at room temperature for 90 minutes. After the reaction wascompleted, the supernatant was discarded, 250 μl/well of a 0.8 mMiodoacetamide solution was added, and the plate was left to stand atroom temperature for 20 hours. Subsequently, the plate was washed with a10 mM phosphate buffer (pH 7.0), and 250 μl/well of a 50 mM Tris-50 mMsodium chloride-1% BSA buffer was added, and the plate was left to standat room temperature for 20 hours to thereby prepare anoligonucleotide-immobilized plate.

Production Example 1-2

[0075] 4.8 mg of anti-AFP antibody clone No. 9, which reacts with AFP,was reacted with 39 μg of pepsin at 37° C. for 30 minutes, and purifiedthrough gel filtration column chromatography to obtain 2.4 mg of anF(ab′)2 fraction. To the obtained fraction, 0.2 M 2-mercaptoethanolaminewas added in an amount {fraction (1/20)} times the volume of the F(ab′)2fraction, to thereby prepare the Fab′ fraction by reduction.

[0076] A full match oligonucleotide having the 5′-terminal modified withan amino linker (manufactured by Amersham Pharmacia Biotech; sequence:5′-AGTGACGAATTCACGGCTGGATCCCTAATG-3′) and a mismatch oligonucleotidehaving the 5′-terminal modified with an amino linker (manufactured byAmersham Pharmacia Biotech; sequence:5′-GATTTTAGCTCTTCTTTGGAGAAAGTGGTG-3′) were respectively dissolved in a50 mM phosphate buffer (pH 7.0), and GMBS dissolved in DMF was added inan amount five times the molar amount of the oligonucleotide. Thereaction was effected at 37° C. for 90 minutes, and the unreacted GMBSwas removed to obtain maleimide-attached oligonucleotides. To each ofthe obtained maleimide-attached oligonucleotide, the Fab′ fraction ofthe anti-AFP antibody was added in an amount 0.2 times the molar amountof the oligonucleotide, reacted, and desalted, to thereby prepare a fullmatch anti-Fab′ oligonucleotide complex (abbreviated as FM complex) anda mismatch anti-Fab′ oligonucleotide complex (abbreviated as MMcomplex).

Example 2-1

[0077] An FM complex solution containing 0.2 wt % Polymer (P1) and an MMcomplex solution containing 0.2 wt % Polymer (P1) were dispensed intothe oligonucleotide-immobilized plate prepared in Production Example 1-1in an amount of 100 μl/well, and incubated at 37° C. for 30 minutes. Theplate was washed with Tris-TritonX (referred to as washing solution (X)hereinbelow), and 100 μl of a 100 ng/ml diluted solution of AFP antigenwas added and incubated at 37° C. for 1 hour. After the plate was washedwith the washing solution (X), 200 μl of an AMPPD solution, achemiluminescent substrate, was added, and incubated at 37° C. for 5minutes. The plate was then read on a fluorescent plate reader ARVOsx(Wallac Beltold) , and the ratio of (luminescence of FMcomplex)/(luminescence of MM complex) was determined from themeasurement results. The results are shown in Table 4.

Examples 2-2 to 2-9 and Comparative Examples 2-1 to 2-3

[0078] Measurements were made following the same procedures as inExample 2-1, except that Polymer (P1) was replaced with each polymershown in Table 4. The results are shown in Table 4.

Comparative Example 2-4

[0079] Measurements were made following the same procedures as inExample 2-1, except that the FM complex solution containing 0.2 wt %Polymer (P1) and the MM complex solution containing 0.2 wt % Polymer(P1) were replaced with the FM and MM complex solutions free of Polymer(P1), respectively. The results are shown in Table 4. TABLE 4 Kind ofPolymer (FM Luminescence)/ (Abbreviation) (MF Luminescence) Example 2-1(P1) 3.2 Example 2-2 (P2) 2.9 Example 2-3 (P3) 12.8 Example 2-4 (P4)44.5 Example 2-5 (P6) 8.4 Example 2-6 (P7) 4.3 Example 2-7 (P8) 3.4Example 2-8 (P9) 6.8 Example 2-9 (P10) 39.1 Comp. Ex. 2-1 (P5) 1.5 Comp.Ex. 2-2 (P11) 1.9 Comp. Ex. 2-3 (P12) 1.4 Comp. Ex. 2-4 — 1.1

[0080] From Table 4, it is understood that the ratios of theluminescence of the FM complex to the luminescence of the MM complex arehigher in Examples than in Comparative Examples. This means that thespecific hybridization was not inhibited but the nonspecifichybridization was inhibited even at 37° C., thereby realizing moreaccurate hybridization.

Example 3-1

[0081] An oligo DNA having the 5′-terminal modified with an amino group(abbreviated as PUC2-NH₂) having the DNA sequence shown below forimmobilization, the PUC having a sequence complementary to PUC2-NH₂, andthe mismatch PUC′ or PUC″ having the 5′-terminal modified with biotin,which are SNPs of PUC, were used. PUC2-NH₂ is manufactured by ESPECOLIGO SERVICE CORP. PUC2-NH₂:5′-(CH₂)₁₂TTTACAACGTCGTGACTGGG-3′

[0082] 100 μl/well of a 25 pmol/ml PUC2-NH₂ solution in E-NaPB wasdispensed into a DNA-BIND 96-well plate (manufactured by CorningIncorporated), and incubated overnight at 4° C. (immobilization ofPUC2-NH₂). Then each well was cleared of the solution, and washed threetimes with D-PBS. 200 μl/well of the B-E-NaPB was added, and incubatedat 37° C. for 1 hour (blocking operation-1). Then each well was clearedof the B-E-NaPB to prepare a PUC2-NH₂-immobilized plate.

[0083] Into the PUC2-NH₂-immobilized plate, 100 μl/well of a 5× SSCsolution containing 3.2 wt % of Polymer (P1) and 0.2 pmol/ml each ofPUC, PUC′, and PUC″ was added, and incubated at 25° C. for 1 hour. Eachwell was cleared of the oligo DNA solution, and washed twice with a 30mM sodium citrate solution containing 0.1% SDS and 300 mM NaCl at 25° C.200 μl/well of the B-E-NaPB was added, and incubated at 37° C. for 30minutes (blocking operation-2), and then each well was cleared of theB-E-NaPB. 100 μl/well of the Avidin-HRP solution was added, andincubated at 37° C. for 30 minutes (avidin-biotin reaction). Each wellwas then washed three times with D-PBS, 100 μl/well of the substratesolution included in the HRP kit was added, and the mixture wasincubated at 25° C. for 10 minutes (POD-substrate reaction).Subsequently, 100 μl/well of the reaction terminator included in the HRPkit was added, and the absorbance at 450 nm was measured by means ofSPECTRA MAX250 (microplate reader, manufactured by MOLECULAR DEVICESCORPORATION). Incidentally, the HRP kit used herein was Coloring Kit Tfor Peroxidase (manufactured by SUMITOMO BAKELITE CO., LTD.).

[0084] From the results of the measurements, the ratios of (absorbanceof PUC)/(absorbance of PUC′) and (absorbance of PUC)/(absorbance ofPUC″) were determined. The results are shown in Table 5.

Examples 3-2 to 3-9 and Comparative Examples 3-1 to 3-2

[0085] Measurements were made following the same procedures as inExample 3-1, except that Polymer (P1) was replaced with each polymershown in Table 5. For Comparative Examples 3-1 and 3-2, only the ratioof (absorbance of PUC)/(absorbance of PUC″) was determined. The resultsare shown in Table 5.

Comparative Example 3-3

[0086] Measurements were made following the same procedures as inExample 3-1, except that the 5× SSC solution containing 3.2 wt % ofPolymer (P1) and 0.2 pmol/ml each of PUC, PUC′, and PUC″ was replacedwith a 5× SSC solution containing 0.2 pmol/ml each of PUC, PUC′, andPUC″. The results are shown in Table 5.

Comparative Example 3-4

[0087] Measurements were made following the same procedures as inExample 3-1, except that the 5× SSC solution containing 3.2 wt % ofPolymer (P1) and 0.2 pmol/ml each of PUC, PUC′, and PUC″ was replacedwith a 5× SSC solution containing 1.0% casein (manufacture bySIGMA-ALDRICH CO.) and 0.1% N-LS (manufactured by WAKO PURE CHEMICALSINDUSTRIES, LTD.). The results are shown in Table 5.

Comparative Example 3-5

[0088] Measurements were made in the same way as in Example 3-1, exceptthat the 5× SSC solution containing 3.2 wt % of Polymer (P1) and 0.2pmol/ml each of PUC, PUC′, and PUC″ was replaced with 0.2 pmol/ml eachof PUC, PUC′, and PUC″, and a 6× SSC solution containing 10 mg/ml Ficol400 (manufactured by Amersham Pharmacia Biotech, trademark), 10 mg/mlpolyvinylpyrrolidone, 10 mg/ml bovine serum albumin, and 0.5% SDS(Denhard's solution). The results are shown in Table 5. TABLE 5 Kind of(Absorbance of (Absorbance of Polymer PUC)/(Absorbance PUC)/(Absorbance(Abbreviation) of PUC′) of PUC″) Example 3-1 (P1) 5.1 2.3 Example 3-2(P2) 4.5 2.9 Example 3-3 (P3) 3.1 2.5 Example 3-4 (P4) 2.7 2.8 Example3-5 (P6) 4.2 3.0 Example 3-6 (P7) 4.0 2.8 Example 3-7 (P8) 3.6 2.6Example 3-8 (P9) 3.4 2.8 Example 3-9 (P10) 3.6 2.8 Comp. Ex. (P11) — 1.83-1 Comp. Ex. (P12) — 2.0 3-2 Comp. Ex. — 2.0 1.0 3-3 Comp. Ex. OtherInhibitor 1.8 1.0 3-4 Comp. Ex. Other Inhibitor 1.6 1.0 3-5

[0089] From Table 5, it is understood that the ratios of the absorbancein full match (absorbance of PUC) to the absorbance in mismatch(absorbance of PUC′ or PUC″) are higher in Examples than in ComparativeExamples. This means that the specific hybridization was not inhibitedbut the nonspecific hybridization was inhibited, thereby realizing moreaccurate hybridization.

What is claimed is:
 1. A nonspecific hybridization inhibitor comprisinga polymer (H) having a nonspecific hybridization inhibitory action, saidpolymer having in its molecule at least one of carboxyl and sulfonegroups, and phosphorylcholine-like groups, and having a weight averagemolecular weight of 1000 to
 5000000. 2. The inhibitor of claim 1,wherein said phosphorylcholine-like group is a group derived from amonomer including a phosphorylcholine-like group represented by theformula (1):

wherein X stands for a divalent organic residue, Y stands for analkyleneoxy group having 1 to 6 carbon atoms, Z stands for a hydrogenatom or R⁵O(C═O)—, provided that R⁵ stands for an alkyl or hydroxyalkylgroup having 1 to 10 carbon atoms; R¹ stands for a hydrogen atom or amethyl group, R², R³, and R⁴ are the same or different groups, and eachstands for a hydrogen atom, or an alkyl or hydroxyalkyl group having 1to 6 carbon atoms; m is 0 or 1, and n denotes an integer of 1 to
 4. 3.The inhibitor of claim 1, wherein said polymer (H) is a polymer obtainedby polymerization of a monomer composition comprising 5 to 95 mol % of amonomer having a phosphorylcholine-like group, 5 to 95 mol % of at leastone of a monomer having a carboxyl group and a monomer having a sulfonegroup.
 4. The inhibitor of claim 1, wherein said polymer (H) is apolymer obtained by polymerization of a monomer composition comprising 5to 90 mol % of a monomer having a phosphorylcholine-like group, 5 to 90mol % of at least one of a monomer having a carboxyl group and a monomerhaving a sulfone group, and 5 to 60 mol % of a hydrophobic monomer. 5.The inhibitor of claim 4, wherein said hydrophobic monomer is a monomerrepresented by the formula (2):

wherein R⁶ stands for a hydrogen atom or a methyl group, L¹ stands for—C₆H₄—, —C₆H₁₀—, —(C═O)O—, —O—, —(C═O)NH—, —O(C═O)—, or —O (C═O)O—, andL² stands for a hydrogen atom, —(CH₂)g-L³, or ((CH₂)_(p)—O)_(h)-L³,wherein g and h each denotes an integer of 1 to 24, p denotes an integerof 3 to 5, and L³ stands for a hydrogen atom, a methyl group, —C₆H₅, or—OC₆H₅.
 6. A clinical diagnostic reagent comprising an inhibitor ofclaim 1 and a test agent.
 7. A method of clinical analysis comprisingthe steps of: (1) contacting a sample with a test agent capable ofhybridizing with a specific nucleic substance in the presence of aninhibitor of claim 1 under particular conditions for hybridizing saidspecific nucleic substance with said test agent, and (2) detecting areactant generated by hybridization with said test agent in step (1). 8.The method of claim 7, wherein in said step (1), an amount of saidinhibitor is such that a content of said polymer (H) of the inhibitor ina hybridization system is 0.0001 to 20 wt %.